The Two Process model of homeostatic sleep regulation posits that the timing of sleep results from an interaction between a circadian process (Process C) and a homeostatically regulated sleep process (Process S). Process C is linked to neural systems that generate circadian rhythms, but the neural basis of Process S is currently unknown. During the previous funding period, we identified a population of GABAergic cells in the cerebral cortex that express neuronal nitric oxide synthase (nNOS) and also express the transcription factor FOS specifically during sleep and after sleep deprivation (SD). Activation of nNOS neurons is negatively correlated with time awake, unrelated to REM sleep, and most closely linked to NREM bout duration and NREM delta energy. In the absence of nNOS, mice have altered slow wave activity (SWA) during both wakefulness and sleep, are unable to sustain long bouts of NREM sleep, and are unable to respond to a homeostatic sleep challenge despite being sleepier than wild type (WT) mice. Based on these results, we hypothesize that cortical nNOS neurons are involved in the homeostatic regulation of sleep and are a neuroanatomical substrate of Process S. To test this hypothesis, we will first ask whether these sleep-active cortical nNOS neurons are projection neurons and use state-of-the-art viral tracers to identify afferents to these neurons. To determine the role of different afferent inputs in regulating corticl nNOS neurons, we will conduct in vitro patch clamp electrophysiology recordings to determine which neurotransmitters and neuromodulators previously implicated in the control of sleep and wakefulness excite or inhibit sleep-active cortical nNOS cells. We will address whether cortical nNOS neurons are causally involved in sleep homeostasis using optogenetic and pharmacogenetic approaches to activate these cells and characterize the effects on sleep/wake architecture and EEG spectra. Lastly, we will assess the role of nitric oxide derived from cortical nNOS neurons in sleep homeostasis by optogenetic and pharmacogenetic stimulation in the presence and absence of nitric oxide inhibitors. These experiments will elucidate the neurobiology of cortical nNOS neurons and allow us to determine whether the activity of cortical sleep-active nNOS cells is indeed related to Process S. The results will not only enhance our understanding of sleep/wake regulation, but may also have implications for understanding the role of sleep in neurological and psychiatric diseases involving the cortex such as epilepsy, anxiety, and schizophrenia.